Research in the current grant period caused us to significantly revise our model of SNARE assembly, resulting in a new and much more specific hypothesis for the fusion mechanism. We now think assembly takes place in a series of well-defined discrete steps, which we call discrete zippering. The new data have come mainly from optical tweezers and biochemical experiments with the isolated proteins showing binary switch assembly behavior. Importantly, this discrete mechanism, unlike continuous zippering, creates discrete assembly intermediates which are natural pivot points for regulation. The discrete zippering concept is helpful as a guide to current research. Our main goal (Aims 1-3 and 5) is to rigorously test the new discrete zipper model to validate or modify it. We will do this by systematically studying the physical chemical, functional, and physiological effects of targeted mutations in each of the discrete portions of the SNARE complex: the N-terminal domain (NTD), C-terminal domain (CTD), linker domain (LD), and the trans-membrane domain (TMD). Many mutations in NTD and CTD are already known that affect fusion physiology in some way (less attention has been paid to TMD and LD) but it is not known how they work molecularly to affect SNARE assembly because sophisticated physical chemical assays have not in general been used before in this connection. Our comprehensive combined physical-chemical and functional mutational analysis of CTD, LD and TMD will provide essential information to advance our understanding of membrane fusion to the next level, whatever the model. Our other goal (Aim 4) is to better understand how the essential gene product Munc18 facilitates the discrete assembly of NTD, inherently a non- physiologically slow process. New data suggest that Munc18 can act as a molecular chaperone to promote the assembly of fusion-competent all-parallel SNARE helical bundles and prevent incompetent anti-parallel arrangements.